FIG 4.

HBZ and CUL1 interact in the cytoplasm. (A) Schematic diagram of the HBZ-NES mutant (HBZ-NES-mut) used in this study. Amino acids altered to alanine are underlined. (B) HEK293T cells were cotransfected with 3 μg of pcDNA3-Myc-CUL1 with (+) or without (−) 3 μg of pcDNA3-HBZ-unspliced form-HA (-US), pcDNA3-HBZ-spliced isoform-HA (-SI), pcDNA3-HBZ-NES-mut-HA (-NES-mut), or pcDNA3-HBZ-NES-mut-PKI-HA [-PKI (NES)]. After 36 h, cell extracts were prepared and subjected to immunoprecipitation (IP) using anti-HA antibody, followed by immunoblotting (IB) with anti-Myc and anti-HA antibodies. Total protein levels in whole-cell lysates (WCL) were analyzed by immunoblotting using anti-Myc, anti-HA, and anti-α-tubulin antibodies.